Phosphorylation is a major signalling mechanism involved in cellular protein regulation. Phospho-specific antibodies that can specifically discriminate between phosphorylated and non-phosphorylated forms of a polypeptide have emerged as key tools for analysis of phosphorylation.
A number of proteins are phosphorylated on histidine, such as NDPK, ATP citrate lyase, the G-protein Gβ subunit, and the potassium channel, KCa3.1, P-selectin and annexin 1. Moreover, these phosphorylation events are linked to a number of diseases including cancer, cardiovascular defects, inflammation and diabetes.
Even though it is believed that 6% of all mammalian phosphorylations occur on histidine, the instability and relative low abundance of phosphohistidine in comparison to phosphoserine and phosphothreonine has made it difficult to study. Similar problems were observed during early studies of phosphotyrosine, due to its relatively low abundance and its instability. However antibodies specific for phosphotyrosine have been developed, allowing the detection of phosphotyrosine residues using immunofluorescence, and for immunoaffinity concentration of the phosphoproteins prior to digestion and mass spectrometric peptide analysis.
The difficulties associated with detection of phosphohistidine residues have led to a number of approaches and techniques in order to understand phosphorylation dynamics of proteins.
The traditional approach of using Edman degradation as a way to identify phosphohistidine residues is possible. However, this approach is not without its limitations as the percentage of protein phosphorylated on phosphohistidine is naturally low, as well as instability of the phosphoramidate bond leading to further decomposition. The acid lability of phosphohistidine is also known to be a limitation in the study of phosphohistidine proteins. Accordingly, the main problem relating to the analysis of phosphohistidine residues is in fact not the detection of the residue, but the techniques used to prepare the samples for analysis. This suggests the main area to concentrate on is the enrichment of complex sample mixtures. In this regard, antibodies appear to have most potential to improve phosphohistidine detection, as they can be utilized in detection and enrichment without the need for acidic conditions.
In recent years a number of phosphospecific antibodies have been developed that can typically detect femtomolar amounts of specific phosphoproteins (Yan, J. X.; Packer, N. H.; Gooley, A. A.; Williams, K. L. J. Chromatogr. A 1998, 808, 23-41). This has led to the development of monoclonal antibodies that recognise phosphoserine, phosphothreonine, and phosphotyrosine residues, via the use of a bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH) conjugate. Each of these antibodies shows no cross reactivity towards other phosphoamino acids, or the respective non-phosphorylated amino acid, however if the residue of interest is buried within the protein the detection of phosphoamino acids can be hindered (Arad-Dann, H.; Beller, U.; Haimovitch, R.; Gavrieli, Y.; Ben-Sasson, S. A. J. Histochem. Cytochem. 1993, 41, 513-9). This means that the selection of antibody needs to be tested against individual proteins.
For phosphoserine and phosphothreonine the generation of antibodies was relatively simple, the antibodies being made by simply conjugating the residues to KLH or BSA. However, this was not the case for phosphotyrosine and phosphohistidine due to them both being unstable under typical biological conditions. Unlike the more commonly studied phosphohydroxyamino acids (phosphoserine, phosphothreonine and phosphotyrosine), the phosphoryl group in phosphohistidine is attached to a histidine ring, as a phosphoramidate bond, which is acid labile making it unstable under physiological conditions and therefore difficult to study. In view of the fact that phosphohistidine is unstable, it cannot be used successfully to generate antibodies.
Due to ease of hydrolysis of phosphohistidine it has been impossible to generate antibodies directly from the amino acid of interest. This has prompted a number of researchers to attempt to design and synthesise a suitable stable analogue of phosphohistidine.
The first report of an antibody that could detect phosphohistidine was by Frackelton and co-workers; when generating a monoclonal antibody towards phosphotyrosine, some cross reactivity was observed for phosphohistidine (Mol. Cell. Biol., 1983, 1343). The first successful approach that targetted phosphohistidine was reported in 2010 by Muir and co-workers (J. Am. Chem. Soc. 2010, 132, 14327), who used phosphoryltriazolylalanine. The phosphoryltriazolylalanine was incorporated into a peptide sequence found in the “tail end” of Histone H4 as a replacement for a specific histidine residue. The peptide was then used to generate antibodies and tested via dot blot analysis. These results demonstrated a selectivity towards the specifically phosphorylated histidine peptide, however the antisera used did not detect the peptide incorporating a phosphohistidine residue at a different site. In addition, further work in this field by Muir and co-workers in 2013 (Nat. Chem. Bio. 2013, 9, 416-421) involved the development of a phosphoryl-triazolylethylamine based polyclonal antibody able to recognize phosphohistidine. However, this particular antibody exhibited high cross reactivity with phosphotyrosine limiting its useful application.
Accordingly, there remains a need for improved antibodies for specifically detecting phosphohistidine or phosphotyrosine.